The proposal by Ehrlich and colleagues, "Molecular Analysis of Pathogens in Otitis Media by PCR," is a non-overlapping continuation research application. The investigations planned will attempt to capitalize on and extend this group's findings dealing with infectious agents in otitis media (OM) currently funded as 5R01 DC02148-03 (Ehrlich), expiration date 11/30/97. Ehrlich's group has documented the presence of bacterial DNA and H. influenzae transcripts in culture-negative, otitis media with effusions (OME). This demonstration is a departure from one theory regarding the etiology of effusion in OM which holds that bacterial products, but not live bacteria, are responsible for causing the effusion. An experimental chinchilla animal model was developed and supported the PCR-based findings and suggested that antibiotic-treated bacteria may persist in a culture-sterile, but PCR-positive state for weeks. This continuing application will concentrate on the molecular biology of H. influenzae and factors which may lead to the development of OME. The central hypothesis which will be explored is whether OME is initiated and maintained by the establishment of biofilms. Biofilms are networks of adherent, or sessile, bacteria distinct from free-floating, or planktonic, forms. Biofilms have been implicated in causing contamination of implantable devices, "sterile" arthritis, and prostatitis. Biofilm formation results from the coordinated expression of sets of genes that are induced upon bacterial attachment. Once formed, multiple species can live in specific microenvironments protected from the external environment and display extraordinary tolerance to antibiotics. This tolerance is primarily attributable to the reduced metabolic activity and lowered rates of cell division of sessile bacteria when compared to planktonic forms. To test these hypotheses the researchers will compare, through differential gene expression, the metabolic activity of free-floating bacteria, biofilm bacteria, and viable, culture-negative organisms from middle-ear effusions. To further characterize the transcriptionally active, effusion-derived H. influenzae, pulse chase experiments using RIPAs will be employed to evaluate levels of protein synthesis in the animal OM model. The focus will also be directed to other infectious agents that have been implicated in OME, including M. catarrhalis and S. pneumoniae, and OME will be assessed for the presence of the corresponding bacterial mRNA species using RT-PCR. In a second series of experiments, the sterile OME-derived H. influenzae mRNAs will be screened for possible transcripts encoding inflammatory mediators. In a third series of experiments, attempts will be made to characterize the genes involved in H. influenzae biofilm formation and compared with the established biofilm-forming agent P. aeruginosa. A combined final series of experiments will attempt to determine the mechanism(s) that regulate or trigger the formation of biofilms and to correlate different models of mutational2 induction with the consequences of antibiotic therapy.